{"gene":"RGS14","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2001,"finding":"The GoLoco motif of RGS14 interacts exclusively with Gαi1, Gαi2, and Gαi3 in their GDP-bound forms and exhibits guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of GDP-to-GTP exchange on Gαi1 and stabilizing the GDP-bound state. This is distinct from the N-terminal RGS box, which confers GTPase-accelerating protein (GAP) activity on Gαi/o subunits, establishing that RGS14 has two independent Gα interaction sites with different biochemical activities.","method":"GTPγS binding assays, intrinsic tryptophan fluorescence assays, in vitro binding experiments with purified proteins","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple in vitro assays (GTPγS binding, fluorescence) with recombinant proteins; replicated across RGS12 and RGS14; foundational biochemical characterization","pmids":["11387333"],"is_preprint":false},{"year":2000,"finding":"RGS14 binds activated Rap1 and Rap2 (but not Ras) through a domain distinct from the RGS domain that shares sequence identity with the Ras/Rap binding domain of B-Raf and Raf-1 kinases. RGS14 preferentially stimulates GTPase activity of Gαo over Gαi1 in brain membranes and in reconstitution assays.","method":"Yeast two-hybrid, in vitro binding with Rap2 mutants, GTPase assays with brain membranes, deletion analysis","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — yeast two-hybrid plus in vitro binding and enzymatic assays in a single study; multiple orthogonal methods","pmids":["10926822"],"is_preprint":false},{"year":2000,"finding":"RGS14 acts as a GAP for Gαi subfamily members and attenuates IL-8 receptor-mediated MAPK activation. RGS14 does not exhibit GAP activity toward Gαs or Gαq. Although not a GAP for G12/13α, RGS14 impairs G13α-mediated c-fos SRE activation. An RGS14 mutant (EN92/93AA) defective in Gαi signaling inhibition still blocks SRE activation. RGS14 localizes predominantly to the cytosol but is recruited to membranes by constitutively active G13αQ226L.","method":"GTPase assays, cell-based signaling reporter assays, subcellular fractionation, dominant-negative mutant analysis","journal":"Molecular pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-based assays plus biochemical GAP assays; single lab but multiple readouts","pmids":["10953050"],"is_preprint":false},{"year":2001,"finding":"Full-length RGS14 in brain exists in two immunoreactive forms (~60 kDa and ~55 kDa) in cytosol and two membrane subpopulations. Recombinant RGS14 is a non-selective GAP for Gαi1 and Gαo, with full-length RGS14 approximately 10-fold more potent than the isolated RGS domain alone. GDI activity toward Gαi (but not Gαo) is restricted to the C-terminus containing the GoLoco domain, and the isolated RBD domain does not act as a GAP for Rap1.","method":"Immunoblotting with affinity-purified antisera, reconstitution GTPase assays, pulldown from brain membranes","journal":"Journal of neurochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution GTPase assays plus domain mapping with isolated fragments; multiple orthogonal approaches in one study","pmids":["11739605"],"is_preprint":false},{"year":2003,"finding":"PKA phosphorylates RGS14 at two specific sites in response to cAMP elevation; one site, threonine 494 (Thr494), is immediately adjacent to the GoLoco motif. Mimicking phosphorylation at Thr494 enhanced GDI activity of RGS14 toward Gαi nearly 3-fold without affecting GAP activity toward Gαi or Gαo.","method":"Radiolabeled phosphate incorporation, PKA inhibitor experiments, phosphomimetic mutant biochemical assays, cAMP stimulation in cells","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assays plus phosphomimetic mutagenesis with functional readout; multiple methods in single study","pmids":["12534294"],"is_preprint":false},{"year":2004,"finding":"The RGS14 GoLoco domain discriminates among Gαi isoforms: it inhibits guanine nucleotide exchange on Gαi1 and Gαi3, but not Gαi2. Gαi2 could be rendered sensitive to RGS14 GDI activity by replacing residues within the αA/αB and αB/αC loops of the α-helical domain, identifying these loops as key selectivity determinants.","method":"In vitro GTPγS binding assays, Gαi2 chimeric mutant analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical assays with mutagenesis identifying structural determinants of selectivity","pmids":["15337739"],"is_preprint":false},{"year":2004,"finding":"RGS14 is expressed in mouse embryos immediately prior to the first mitosis, colocalizes with the anastral mitotic apparatus of the mouse zygote, and is essential for completion of the first mitotic division. Loss of Rgs14 in the zygote causes cytofragmentation and failure to progress to the 2-cell stage. During interphase in proliferating cells RGS14 segregates to the nucleus, and during mitosis it localizes to the mitotic spindle and centrioles. Altering RGS14 levels in proliferating cells causes growth arrest.","method":"Loss-of-function in mouse embryos, colocalization imaging, overexpression/knockdown in proliferating cells","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic loss-of-function with clear cellular phenotype, direct localization imaging; replicated in multiple experimental systems","pmids":["15525537"],"is_preprint":false},{"year":2004,"finding":"The RGS and GoLoco domains of RGS14 are independently able to inhibit signaling downstream of Gi via the βγ pathway. Targeting the isolated GoLoco domain to membranes (by myristoylation/palmitoylation or Rap binding) enhances its inhibitory activity. In the context of full-length RGS14, the RGS and GoLoco domains cooperate to confer maximal inhibition of Gi signaling.","method":"Cell-based signaling assays in HEK-m2 cells with domain mutants and truncation constructs, myristoylation targeting constructs","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-based functional assays with domain mutants and multiple constructs; single lab","pmids":["15112653"],"is_preprint":false},{"year":2004,"finding":"RGS14 localizes to centrosomes via its first Ras-binding domain, and shuttles between cytoplasm and nucleus via CRM-1-dependent nuclear export. Mutation of the nuclear export signal or leptomycin B treatment causes nuclear accumulation and association with PML nuclear bodies. A mutant defective in nuclear export also fails to target to centrosomes, indicating that nucleocytoplasmic shuttling is required for centrosomal localization.","method":"Immunofluorescence, leptomycin B treatment, NES mutant analysis, domain deletion constructs","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiments with domain mapping and pharmacological perturbation; single lab","pmids":["15520006"],"is_preprint":false},{"year":2005,"finding":"RGS14 is a microtubule-associated protein that binds directly to polymerized (but not depolymerized) microtubules with Kd ~1.3 μM. RGS14 copurifies with tubulin through multiple polymerization/depolymerization cycles from brain. Both RGS14 and Gαi1-GTP promote tubulin polymerization in the presence of microtubule-associated proteins, but preincubation of RGS14 with Gαi1-GDP abolishes this activity. Depletion of RGS14 from cell extracts blocks mitotic aster formation in vitro.","method":"Microtubule co-sedimentation, direct binding to pure tubulin with Kd measurement, brain extract purification, in vitro aster formation assay, RGS14 depletion","journal":"Cell cycle (Georgetown, Tex.)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified proteins, direct binding Kd measurement, functional depletion assay; multiple orthogonal methods","pmids":["15917656"],"is_preprint":false},{"year":2005,"finding":"The RBD/GoLoco region of RGS14 (lacking the RGS domain) binds Gαo-GDP, Gαi-GDP, and Gβγ in brain pull-down assays. In reconstituted receptor-G protein systems, this region potentiates RGS4 GAP activity by increasing the apparent affinity of RGS4 for Gαi and Gαo, an activity requiring membranes and/or receptors.","method":"Brain pull-down assays, reconstituted M2 receptor/G-protein GTPase assays, steady-state GTPase measurements","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro biochemical assays with brain membranes and reconstitution; multiple activity measurements in single lab","pmids":["15807543"],"is_preprint":false},{"year":2006,"finding":"RGS14 subcellular localization in HeLa cells is controlled by distinct domains: localization to the nucleus requires the RGS and RBD domains; translocation out of the nucleus depends on the GoLoco/GPR domain; localization to centrosomes depends on the RBD domain. RGS14 binds directly to both inactive (GDP) and active (GTP) forms of Gαi1 and Gαi3, but not Gαi2, and binding to inactive Gαi1/3 at the plasma membrane prevents nuclear or centrosomal association.","method":"Confocal imaging of GFP-tagged constructs with domain mutations, co-immunoprecipitation with Gαi isoforms from cells","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct imaging with domain mutants plus co-IP with different Gα isoforms; single lab, multiple readouts","pmids":["16870394"],"is_preprint":false},{"year":2006,"finding":"RGS14 GAP activity and GDI activity toward heterotrimeric G proteins are unaffected by Rap1/Rap2 binding. A heterotrimer composed of N-myristoylated Gαi1 and prenylated Gβγ is resistant to the GDI activity of the RGS14 GoLoco domain, suggesting RGS14 alone cannot dissociate intact heterotrimers.","method":"In vitro GTPase assays, GDI assays with assembled heterotrimer, Rap-binding competition experiments","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical assays; negative finding confirmed across multiple assay conditions in single lab","pmids":["16246175"],"is_preprint":false},{"year":2009,"finding":"In cellular contexts, RGS14 selectively binds activated H-Ras-GTP (not Rap isoforms), assembles a multiprotein complex with components of the ERK MAPK pathway (Raf, MEK, ERK) in an H-Ras-GTP-dependent manner, and promotes sustained ERK activation. siRNA knockdown of RGS14 inhibited NGF- and bFGF-mediated neuronal differentiation of PC12 cells, a process dependent on Ras-ERK signaling.","method":"Co-immunoprecipitation, siRNA knockdown, PC12 neurite outgrowth assays, ERK phosphorylation assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP and loss-of-function with specific cellular phenotype; single lab, multiple methods","pmids":["19319189"],"is_preprint":false},{"year":2010,"finding":"RGS14 is highly enriched in CA2 pyramidal neurons and suppresses long-term potentiation (LTP) at Schaffer collateral synapses. Deletion of RGS14 (exons 2–7 knockout) enables robust LTP at CA2 synapses without affecting CA1 plasticity. MEK inhibition blocks LTP in RGS14-deficient CA2 neurons, implicating ERK/MAP kinase signaling. RGS14 KO mice show enhanced spatial learning and object recognition memory without deficits in non-hippocampal tasks.","method":"Genetic knockout mice, electrophysiology (LTP recordings), MEK inhibitor pharmacology, behavioral testing","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with defined LTP phenotype, pharmacological rescue with MEK inhibitor, behavioral validation; multiple orthogonal methods","pmids":["20837545"],"is_preprint":false},{"year":2010,"finding":"Activated H-Ras binds RGS14 at the R1 Ras-binding domain (RBD) to form a stable complex at cell membranes. RGS14 co-localizes with and forms a complex with Raf kinases in cells, and the regulatory region of Raf-1 binds the RBD region of RGS14. H-Ras and Raf facilitate one another's binding to RGS14. RGS14 selectively inhibits PDGF- but not EGF- or serum-stimulated ERK phosphorylation, dependent on H-Ras binding. Gαi1 binding to RGS14 is mutually exclusive with Raf binding, and Gαi1 recruits RGS14 to the plasma membrane to reverse inhibition of ERK signaling.","method":"Co-immunoprecipitation, subcellular localization imaging, ERK phosphorylation assays, domain mutant analysis","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus functional signaling assays; single lab, multiple methods","pmids":["19878719"],"is_preprint":false},{"year":2011,"finding":"RGS14 forms a Gαi/o-dependent complex with the α2A-adrenergic receptor (α2A-AR) in live cells, as measured by BRET. Receptor agonist (UK14304) stimulation disrupts the RGS14·α2A-AR complex while preserving the RGS14·Gαi1 complex. Ric-8A (a non-receptor GEF) facilitates dissociation of both the RGS14·Gαi1 complex and the Gαi1-dependent RGS14·α2A-AR complex after receptor activation.","method":"Bioluminescence resonance energy transfer (BRET) in live cells, pharmacological receptor stimulation","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — BRET in live cells with receptor agonist and genetic perturbations; single lab, single primary method","pmids":["21880739"],"is_preprint":false},{"year":2012,"finding":"RGS14 preferentially binds activated H-Ras-GTP in live cells, and inactive Gαi1-GDP markedly enhances RGS14 affinity for H-Ras-GTP (but active Gαi1-GTP does not). Active H-Ras-GTP interacts with a native RGS14·Gαi1 complex in brain lysates. Co-expression of RGS14 and Gαi1 in PC12 cells greatly enhances H-Ras-stimulatory effects on neurite outgrowth. Stimulation of α2A-AR induces a conformational change in the Gαi1·RGS14·H-Ras ternary complex.","method":"BRET in live cells, co-immunoprecipitation from brain lysates, PC12 neurite outgrowth assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — BRET, brain co-IP, and functional neurite outgrowth assay; multiple orthogonal methods in single study","pmids":["23250758"],"is_preprint":false},{"year":2013,"finding":"The RBD region of RGS14 mediates an intramolecular interaction that enhances GAP activity of the RGS domain while simultaneously inhibiting GDI activity of the GPSM/GoLoco domain. The RBD region also binds members of the B/R4 subfamily of RGS proteins and enhances their effects on GPCR-activated Gi/o proteins.","method":"In vitro GTPase/GDI assays with isolated domains and full-length protein, protein-protein binding assays","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro biochemical assays; single lab, novel intramolecular regulatory mechanism","pmids":["23255434"],"is_preprint":false},{"year":2015,"finding":"RGS14 forms a stable complex with inactive Gαi1-GDP at the plasma membrane via its GPR motif and is also recruited to the plasma membrane by activated Gαo-AlF4(-) via its RGS domain. RGS14 undergoes allosteric conformational changes upon Gαi1-GDP binding to the GPR motif, as revealed by hydrogen/deuterium exchange mass spectrometry. RGS14 can form a ternary complex with Gαo-AlF4(-) and a Gαi1-GDP mutant (G42R) simultaneously, and a preformed RGS14·Gαi1-GDP complex retains full capacity to stimulate GTPase activity of Gαo-GTP.","method":"Size exclusion chromatography, hydrogen/deuterium exchange mass spectrometry (HDX-MS), BRET, biochemical reconstitution, cellular localization assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — HDX-MS structural approach combined with reconstitution of ternary complex and functional GTPase assay; multiple orthogonal methods","pmids":["25666614"],"is_preprint":false},{"year":2016,"finding":"RGS14 acts as a dedicated GAP for Gαo/i signaling and does not prolong Gβγ signaling following receptor activation. Co-expression of RGS14 inhibits free Gβγ release after agonist stimulation and increases the deactivation rate of Gα, consistent with GAP activity. Overexpression of inactive Gαi1 to recruit RGS14 to the plasma membrane did not alter RGS14's GAP activity toward a second Gαo protein, indicating the GPR motif functions independently of the RGS domain for GAP activity.","method":"BRET in live cells, GPCR agonist/antagonist kinetics analysis","journal":"Pharmacology research & perspectives","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — BRET live cell assays measuring both Gα and Gβγ dynamics; single lab, single method","pmids":["27713821"],"is_preprint":false},{"year":2017,"finding":"Endogenous RGS14 in neuroblastoma (B35) cells localizes to juxtanuclear membranes encircling the nucleus, at nuclear pore complexes on both sides of the nuclear envelope and within intranuclear membrane channels, and within chromatin-rich and chromatin-poor nuclear regions in a cell-cycle-dependent manner. Endogenous RGS14 was absent from the plasma membrane in resting cells but could be trafficked to the plasma membrane from juxtanuclear membranes (derived from ER/Golgi) following AlF4¯ activation of its G protein binding partners.","method":"Confocal imaging, 3D-structured illumination microscopy, AlF4¯ pharmacological activation, subcellular fractionation","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — super-resolution and confocal imaging of endogenous protein; functional trafficking experiment; single lab","pmids":["28934222"],"is_preprint":false},{"year":2018,"finding":"RGS14 directly interacts with Ca2+/calmodulin (CaM) and is phosphorylated by CaMKII in vitro. RGS14 associates with CaMKII and CaM in hippocampal CA2 neurons in vivo. These interactions were identified via immunoprecipitation-mass spectrometry proteomics of endogenous RGS14 from mouse brain, validated by direct biochemical binding assays.","method":"Immunoprecipitation-mass spectrometry from mouse brain, direct binding assays, in vitro CaMKII phosphorylation assay, confocal colocalization","journal":"Journal of proteome research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomics discovery validated by in vitro assays and co-IP; multiple methods in single lab","pmids":["29518331"],"is_preprint":false},{"year":2018,"finding":"RGS14 suppresses plasticity in hippocampal area CA2 by restricting calcium (Ca2+) elevations in dendritic spines. Genetic KO of RGS14 results in nascent LTP requiring NMDA receptors, CaMK, and PKA. WT CA2 neurons display significantly attenuated spine Ca2+ transients during structural plasticity induction compared with RGS14 KO CA2 or CA1 controls. Acute overexpression of RGS14 is sufficient to block spine structural plasticity, and elevated extracellular Ca2+ restores plasticity to RGS14-expressing neurons.","method":"Genetic KO mice, two-photon calcium imaging in dendritic spines, pharmacological inhibition (NMDA receptor, CaMK, PKA antagonists), overexpression experiments","journal":"eNeuro","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct spine Ca2+ imaging combined with KO and OE experiments plus pharmacological dissection; multiple orthogonal methods","pmids":["29911178"],"is_preprint":false},{"year":2018,"finding":"14-3-3γ directly binds RGS14 at two distinct sites: one phosphorylation-independent site and one phosphorylation-dependent site at Ser-218, potentiated by active H-Ras signaling. The pSer-218-dependent interaction inhibits active Gαi1-AlF4- binding to the RGS domain but does not affect H-Ras or inactive Gαi1-GDP binding. The phosphorylation-independent 14-3-3 interaction inhibits RGS14 nuclear import/nucleocytoplasmic shuttling without affecting Gαi interactions.","method":"BRET in live cells, direct binding assays, site-directed mutagenesis at Ser-218","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — BRET plus mutagenesis identifying two functionally distinct binding sites; single lab, multiple readouts","pmids":["30093406"],"is_preprint":false},{"year":2020,"finding":"Human genetic variants L505R (LR) and R507Q (RQ) in the nuclear export sequence of RGS14 disrupt RGS14 binding to Gαi1-GDP and to Exportin 1 (XPO1), disturb nucleocytoplasmic equilibrium, and abolish RGS14's capacity to inhibit LTP. Variant LR accumulates irreversibly in the nucleus, preventing membrane localization to dendritic spines. When introduced into mice by CRISPR/Cas9, RGS14-LR is predominantly nuclear in neurons of hippocampus, amygdala, piriform cortex, and striatum. Unlike complete RGS14 KO, LR mice show normal spatial learning, suggesting distinct nuclear vs. dendritic spine functions.","method":"Site-directed mutagenesis, co-immunoprecipitation, electrophysiology (LTP), CRISPR/Cas9 knock-in mice, confocal imaging, behavioral testing","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP for binding partners, LTP electrophysiology, CRISPR knock-in mice, behavioral phenotyping; multiple orthogonal methods in single study","pmids":["33410399"],"is_preprint":false},{"year":2022,"finding":"RGS14 possesses a carboxy-terminal PDZ ligand required for binding to NHERF1 scaffold protein (at PDZ2) and thereby regulates NPT2A-mediated renal phosphate transport. RGS14 blocks PTH- and FGF23-induced dissociation of the NPT2A-NHERF1 complex and thereby suppresses hormone-sensitive (but not basal) phosphate uptake. RGS14 genetic variants bearing mutations in the PDZ ligand disrupt NHERF1 binding and PTH-sensitive phosphate transport. RGS14 does not affect PTH-directed G protein activation or cAMP formation, implying a post-receptor site of action.","method":"Isotope uptake measurements, BRET assays, siRNA knockdown, pull-down and overlay assays, molecular modeling, direct binding assays in proximal tubule cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (isotope transport, BRET, pull-down, mutagenesis, siRNA) in single study","pmids":["35307350"],"is_preprint":false},{"year":2007,"finding":"RGS14 associates with mu-opioid receptors (MOR) in periaqueductal gray (PAG) neurons. When RGS14 is silenced, morphine increases serine 375 phosphorylation of MOR (a GRK substrate), leading to receptor internalization and recycling and resensitization to morphine with reduced tolerance. RGS14 prevents GRKs from phosphorylating MOR residues required for β-arrestin-mediated endocytosis. Morphine (but not DAMGO) also triggers CaMKII activation in PAG neurons via a mechanism requiring RGS14.","method":"siRNA knockdown in vivo, receptor phosphorylation assays, internalization/recycling assays, behavioral antinociception measurement","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo knockdown with mechanistic readouts of receptor phosphorylation and internalization; single lab","pmids":["17825524"],"is_preprint":false},{"year":2013,"finding":"RGS14 regulates αMβ2 integrin activation during phagocytosis in macrophages. Exogenous RGS14 expression in COS-7 cells increases binding of C3bi-opsonized targets. Knockdown of RGS14 in J774.A1 macrophages decreases association with C3bi-opsonized particles. This function requires the R333 residue of the RGS14 RBD and the F754 residue of β2, indicating regulation occurs through the Rap/RBD axis.","method":"Exogenous expression, siRNA knockdown, binding assays with opsonized particles, point mutant analysis","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function experiments with mutant analysis; single lab, multiple cell types","pmids":["23805333"],"is_preprint":false},{"year":2023,"finding":"RGS14 localizes to mitochondria in hippocampal CA2 pyramidal cells and reduces mitochondrial respiration in vitro. Loss of RGS14 dramatically increases 3-nitrotyrosine levels (oxidative stress marker) in CA2 cells, which is exacerbated following kainic acid-induced status epilepticus and correlates with a lack of SOD2 induction. RGS14 KO mice show accelerated onset of limbic motor seizures and increased mortality. RGS14 KO also results in absence of microgliosis in CA1 and CA2 following seizure.","method":"Mitochondrial localization imaging, in vitro mitochondrial respiration assay, kainic acid seizure model in KO mice, proteomics, immunostaining for oxidative stress markers","journal":"Neurobiology of disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mice with defined seizure phenotype, localization experiment, in vitro functional assay; single lab, multiple methods","pmids":["37075948"],"is_preprint":false},{"year":2025,"finding":"Small-molecule inhibitors targeting the solvent-exposed 'canyon' in the RGS14 RGS-box that interacts with the Gα switch I region can non-covalently and selectively inhibit RGS14 GAP activity. Two second-generation analogs (Z55660043 and Z55627844) inhibited RGS14 GAP activity in both fluorescence-based and radioactive GTP hydrolysis assays without measurable cytotoxicity. One analog showed favorable in vivo pharmacokinetics and CNS penetration.","method":"Structure-guided virtual screening, fluorescence-based GAP assays, radioactive GTP hydrolysis assays, cytotoxicity assays, in vivo pharmacokinetics","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro GAP assays with confirmed inhibition by two orthogonal methods; preprint, single lab, no structural validation yet","pmids":["bio_10.1101_2025.06.11.659181"],"is_preprint":true},{"year":2025,"finding":"RGS14 physically interacts with GNAI3 (Gαi3) in spermatogonial stem cells (SSCs), as shown by co-immunoprecipitation. RGS14 knockdown suppresses SSC proliferation, induces apoptosis, inhibits PLPP2 expression, and reduces MAPK signaling activation. Overexpression of PLPP2 rescues phenotypic defects caused by RGS14 depletion, identifying a RGS14–GNAI3–PLPP2 regulatory axis.","method":"Single-cell RNA sequencing, siRNA knockdown, co-immunoprecipitation, RNA sequencing, PLPP2 rescue overexpression, proliferation/apoptosis assays","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus loss-of-function with rescue experiment; single lab, multiple methods","pmids":["40352663"],"is_preprint":false},{"year":2025,"finding":"RGS14 regulates hormone-sensitive renal phosphate transport through a linker region between the RGS and RBD1 domains: Ser266 and Ser269 within this α-helical linker are phosphorylated in response to PTH and FGF23. Ser266Ala/Ser269Ala substitutions abolish RGS14 regulatory effects on hormone-sensitive phosphate transport while an intact PDZ ligand is also required. Truncation constructs lacking the RGS domain are fully functional for phosphate transport regulation.","method":"RGS14 truncation constructs, phosphorylation assays with PTH and FGF23, phosphate uptake measurements, alanine mutagenesis of Ser266/Ser269","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — mutagenesis combined with functional transport assays and direct phosphorylation measurement; single lab","pmids":["39792018"],"is_preprint":false}],"current_model":"RGS14 is a multifunctional scaffolding protein that integrates heterotrimeric G protein and Ras/Raf/ERK MAPK signaling: its N-terminal RGS domain acts as a GAP for Gαi/o-GTP, its C-terminal GoLoco/GPR motif binds inactive Gαi1/3-GDP to exert GDI activity (potentiated by PKA phosphorylation at Thr494), and its tandem Ras-binding domains selectively bind activated H-Ras-GTP in cells to scaffold a Ras–Raf–MEK–ERK complex; inactive Gαi1-GDP binding enhances H-Ras affinity and recruits the full complex to the plasma membrane while displacing Raf, allowing GPCRs to dynamically regulate the complex; in hippocampal CA2 neurons RGS14 suppresses LTP and learning by restricting spine Ca2+ elevations and downstream CaMK/PKA/ERK signaling; RGS14 also localizes to mitochondria, centrosomes, the mitotic spindle, and the nucleus (via CRM1-dependent export), directly binds polymerized microtubules to promote spindle assembly, interacts with 14-3-3γ to regulate both its Gα-binding and nuclear import functions, and regulates renal phosphate homeostasis through a PDZ-ligand/NHERF1/NPT2A axis that is controlled by phosphorylation of linker-region serines in response to PTH and FGF23."},"narrative":{"mechanistic_narrative":"RGS14 is a multidomain scaffolding protein that integrates heterotrimeric G protein signaling with Ras/Raf/ERK MAPK signaling and microtubule-based mitotic functions [PMID:11387333, PMID:19319189]. Its N-terminal RGS domain accelerates GTP hydrolysis on Gαi/o subunits, while a C-terminal GoLoco/GPR motif binds Gαi1 and Gαi3 (but not Gαi2) selectively in their GDP-bound state and acts as a guanine nucleotide dissociation inhibitor, establishing two independent, biochemically distinct Gα interaction sites [PMID:11387333, PMID:11739605, PMID:15337739]. These activities are regulated post-translationally: PKA phosphorylation at Thr494 adjacent to the GoLoco motif enhances GDI activity ~3-fold, and an intramolecular interaction mediated by the Ras-binding region enhances GAP activity while inhibiting GDI activity [PMID:12534294, PMID:23255434]. Through its tandem Ras-binding domains RGS14 selectively binds activated H-Ras-GTP in cells and assembles an H-Ras-dependent Raf–MEK–ERK complex to promote sustained ERK activation and Ras-dependent neuronal differentiation, with inactive Gαi1-GDP markedly enhancing H-Ras affinity and Gαi1 binding being mutually exclusive with Raf binding, allowing GPCRs to dynamically tune the complex [PMID:19319189, PMID:19878719, PMID:23250758]. RGS14 shuttles between cytoplasm and nucleus via CRM1/XPO1-dependent export and localizes to centrosomes, the mitotic spindle, and microtubules, binding polymerized microtubules directly to promote spindle/aster assembly and being required for the first embryonic mitotic division [PMID:15525537, PMID:15520006, PMID:15917656]. In hippocampal CA2 pyramidal neurons RGS14 suppresses long-term potentiation and spatial learning by restricting dendritic spine Ca2+ elevations and downstream NMDA-receptor/CaMK/PKA/ERK signaling [PMID:20837545, PMID:29911178]. RGS14 also regulates hormone-sensitive renal phosphate transport through a C-terminal PDZ ligand that binds NHERF1 to stabilize the NPT2A–NHERF1 complex against PTH/FGF23-induced dissociation, an activity controlled by phosphorylation of linker serines Ser266/Ser269 [PMID:35307350, PMID:39792018]. Human variants in the RGS14 nuclear export sequence (L505R, R507Q) disrupt Gαi1 and XPO1 binding, trap the protein in the nucleus, and abolish its capacity to inhibit LTP [PMID:33410399].","teleology":[{"year":2000,"claim":"Established that RGS14 acts on G protein signaling through a GAP activity selective for the Gαi/o subfamily and through a domain binding activated Rap1/Rap2, defining it as more than a simple GTPase regulator.","evidence":"GTPase assays with brain membranes, yeast two-hybrid, and cell-based SRE reporter assays","pmids":["10926822","10953050"],"confidence":"High","gaps":["Did not resolve whether Rap binding is functional in cells","Mechanism linking RGS14 to MAPK attenuation unclear"]},{"year":2001,"claim":"Resolved that RGS14 carries two independent Gα interaction sites with opposite biochemical activities — an RGS-box GAP and a GoLoco-motif GDI selective for GDP-bound Gαi1/2/3 — defining its bifunctional regulation of the Gαi cycle.","evidence":"GTPγS binding and intrinsic tryptophan fluorescence assays with purified proteins, plus reconstitution GTPase assays and domain mapping","pmids":["11387333","11739605"],"confidence":"High","gaps":["Cellular consequences of dual GAP/GDI activity not addressed","No structural model of the two binding modes"]},{"year":2003,"claim":"Showed that PKA phosphorylation at Thr494 adjacent to the GoLoco motif selectively potentiates GDI activity, providing a mechanism by which cAMP signaling tunes RGS14's effect on Gαi.","evidence":"In vitro PKA kinase assays, phosphomimetic mutagenesis with GDI/GAP readouts, cAMP stimulation in cells","pmids":["12534294"],"confidence":"High","gaps":["Identity of second PKA site not functionally defined","In vivo relevance of Thr494 phosphorylation untested"]},{"year":2004,"claim":"Linked RGS14 to cell division and subcellular trafficking, showing it is essential for the first embryonic mitosis, localizes to centrosomes/spindle via its RBD, and shuttles between nucleus and cytoplasm via CRM1-dependent export.","evidence":"Loss-of-function in mouse zygotes, colocalization imaging, NES mutagenesis with leptomycin B, domain deletion constructs","pmids":["15525537","15520006"],"confidence":"High","gaps":["Molecular role of RGS14 at the spindle not defined","Link between G protein activities and mitotic function unclear"]},{"year":2005,"claim":"Identified RGS14 as a direct microtubule-associated protein that binds polymerized microtubules and promotes tubulin polymerization and aster formation, providing a biochemical basis for its spindle role.","evidence":"Microtubule co-sedimentation, direct Kd measurement with pure tubulin, brain extract co-purification, in vitro aster formation with RGS14 depletion","pmids":["15917656"],"confidence":"High","gaps":["How Gαi1-GDP inhibition of tubulin polymerization is regulated in vivo unknown","Mitotic phenotype mechanism not connected to MT binding directly"]},{"year":2006,"claim":"Mapped the domain logic of RGS14 localization, showing nuclear targeting requires RGS+RBD, nuclear exit requires GoLoco, and centrosome targeting requires the RBD, while inactive Gαi1/3 binding at the membrane sequesters RGS14 away from nucleus/centrosome.","evidence":"Confocal imaging of GFP-tagged domain mutants and co-IP with Gαi isoforms","pmids":["16870394","16246175"],"confidence":"Medium","gaps":["Single-lab imaging not independently confirmed","Whether RGS14 dissociates intact heterotrimers in cells unresolved"]},{"year":2009,"claim":"Established RGS14 as a scaffold for the Ras–Raf–MEK–ERK pathway, showing it selectively binds H-Ras-GTP in cells, assembles an ERK-pathway complex, and is required for Ras-dependent neuronal differentiation.","evidence":"Co-IP, siRNA knockdown, PC12 neurite outgrowth and ERK phosphorylation assays","pmids":["19319189"],"confidence":"Medium","gaps":["Direct vs. indirect Raf/MEK/ERK binding not fully dissected","Single cellular system"]},{"year":2010,"claim":"Defined the architecture and regulation of the RGS14–H-Ras–Raf complex, showing H-Ras binds the R1 RBD, H-Ras and Raf cooperatively bind RGS14, and Gαi1 binding is mutually exclusive with Raf, letting Gαi1 reverse RGS14's ERK inhibition by membrane recruitment; in parallel, in vivo work revealed RGS14 suppresses CA2 LTP and learning via ERK.","evidence":"Co-IP, localization imaging, ERK assays with domain mutants; and RGS14 KO mice with LTP electrophysiology, MEK inhibitor rescue, and behavioral testing","pmids":["19878719","20837545"],"confidence":"High","gaps":["How GPCR input switches the complex in neurons not directly shown","CA2-specific molecular targets of RGS14 not fully defined"]},{"year":2011,"claim":"Demonstrated that RGS14 forms a Gαi/o-dependent complex with the α2A-adrenergic receptor that is disrupted by agonist and by the GEF Ric-8A, placing RGS14 in a receptor-coupled signaling module.","evidence":"BRET in live cells with pharmacological receptor stimulation and Ric-8A co-expression","pmids":["21880739"],"confidence":"Medium","gaps":["Direct vs. Gα-bridged receptor contact not distinguished","Single primary method"]},{"year":2012,"claim":"Showed that inactive Gαi1-GDP allosterically enhances RGS14 affinity for H-Ras-GTP and that a native Gαi1·RGS14·H-Ras ternary complex exists in brain and is conformationally responsive to α2A-AR stimulation, unifying G protein and Ras inputs on one scaffold.","evidence":"BRET in live cells, co-IP from brain lysates, PC12 neurite outgrowth assays","pmids":["23250758"],"confidence":"High","gaps":["Structural basis of Gαi-enhanced H-Ras binding unknown","Downstream output of the ternary complex in neurons not measured"]},{"year":2013,"claim":"Revealed an intramolecular RBD-mediated regulation that reciprocally enhances RGS-domain GAP activity while inhibiting GoLoco GDI activity, and showed RGS14 binds and potentiates B/R4 RGS proteins, adding autoregulatory and cooperative layers.","evidence":"In vitro GTPase/GDI assays with isolated and full-length constructs and protein-protein binding assays; and macrophage integrin/phagocytosis assays","pmids":["23255434","23805333"],"confidence":"Medium","gaps":["In vivo significance of intramolecular regulation untested","Phagocytosis role from single lab"]},{"year":2015,"claim":"Captured the conformational logic of G protein engagement, showing GPR-bound Gαi1-GDP induces allosteric changes and that RGS14 can simultaneously bind Gαi1-GDP (GPR) and Gαo (RGS) while retaining GAP activity, establishing functional independence of the two Gα sites.","evidence":"Size exclusion chromatography, HDX-MS, BRET, biochemical reconstitution of the ternary complex with GTPase assay","pmids":["25666614"],"confidence":"High","gaps":["No high-resolution crystal/cryo-EM structure","How allostery couples to Ras/ERK scaffolding not shown"]},{"year":2017,"claim":"Localized endogenous RGS14 to juxtanuclear ER/Golgi-derived membranes, nuclear pore complexes, and intranuclear channels, and showed it can traffic to the plasma membrane upon G protein activation, refining its dynamic localization in neuronal cells.","evidence":"Confocal and 3D-SIM super-resolution imaging of endogenous protein with AlF4⁻ activation and fractionation","pmids":["28934222"],"confidence":"Medium","gaps":["Functional role at nuclear pores undefined","Single-lab imaging"]},{"year":2018,"claim":"Connected RGS14 to Ca2+/CaMKII signaling and 14-3-3γ regulation, showing RGS14 binds CaM, is a CaMKII substrate, and is regulated by dual 14-3-3γ sites that separately control Gαi binding and nuclear shuttling, while its CA2 LTP suppression operates by limiting spine Ca2+.","evidence":"IP-mass spectrometry from brain, in vitro binding and CaMKII phosphorylation, BRET, Ser-218 mutagenesis; and two-photon spine Ca2+ imaging with KO/OE and pharmacology","pmids":["29518331","29911178","30093406"],"confidence":"High","gaps":["How CaMKII phosphorylation feeds back on RGS14 function not resolved","Link between 14-3-3γ regulation and spine Ca2+ control untested"]},{"year":2020,"claim":"Demonstrated that human RGS14 NES variants (L505R, R507Q) disrupt Gαi1 and XPO1 binding, trap RGS14 in the nucleus, and abolish LTP inhibition, while leaving spatial learning intact, dissociating RGS14's nuclear from dendritic-spine functions.","evidence":"Site-directed mutagenesis, co-IP, LTP electrophysiology, CRISPR/Cas9 knock-in mice, imaging, behavior","pmids":["33410399"],"confidence":"High","gaps":["Nuclear function of RGS14 not mechanistically defined","Disease association of variants not established"]},{"year":2022,"claim":"Established a non-canonical, post-receptor renal function in which RGS14's C-terminal PDZ ligand binds NHERF1 to stabilize the NPT2A–NHERF1 complex and suppress PTH/FGF23-sensitive phosphate uptake, independent of G protein/cAMP signaling.","evidence":"Isotope phosphate uptake, BRET, siRNA, pull-down/overlay assays, mutagenesis in proximal tubule cells","pmids":["35307350"],"confidence":"High","gaps":["In vivo whole-animal phosphate phenotype not shown","Whether other RGS14 domains modulate this axis unclear"]},{"year":2023,"claim":"Identified a mitochondrial role for RGS14 in CA2 neurons, where it reduces respiration and limits oxidative stress, with KO mice showing accelerated seizures and increased mortality, linking RGS14 to neuronal metabolic and neuroprotective functions.","evidence":"Mitochondrial localization imaging, respiration assays, kainic acid seizure model in KO mice, proteomics, oxidative stress immunostaining","pmids":["37075948"],"confidence":"Medium","gaps":["Molecular mechanism of respiration control unknown","Whether mitochondrial role depends on G protein/Ras activities unclear"]},{"year":2025,"claim":"Refined the renal mechanism and extended RGS14 into spermatogonial stem cell biology and pharmacology: linker serines Ser266/Ser269 are PTH/FGF23-phosphorylated and required for phosphate-transport regulation; RGS14 binds GNAI3 to drive an SSC proliferation/MAPK/PLPP2 axis; and selective small-molecule RGS-box inhibitors were developed.","evidence":"Truncation/alanine mutagenesis with phosphate transport and phosphorylation assays; co-IP, siRNA, RNA-seq and PLPP2 rescue in SSCs; structure-guided GAP-inhibitor screening (one preprint)","pmids":["39792018","40352663","bio_10.1101_2025.06.11.659181"],"confidence":"Medium","gaps":["Kinase responsible for Ser266/Ser269 phosphorylation not identified","Inhibitors lack structural validation and in vivo efficacy data","SSC axis from single lab"]},{"year":null,"claim":"How RGS14's distinct activities — Gαi/o GAP/GDI, H-Ras/ERK scaffolding, microtubule binding, mitochondrial regulation, nuclear shuttling, and renal NHERF1/NPT2A control — are coordinately switched within a single cell, and what defines the nuclear function, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated structural model coupling the multiple domains","Nuclear function of RGS14 undefined","Physiological switch governing which activity dominates in a given cell type unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,2,3,4,5,12,18,20]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[13,15,17,26]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[9]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[9,6]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,3,11]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[11,15,19,21]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6,8,11,21,25]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[6,8,11]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[9]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[29]},{"term_id":"GO:0005635","term_label":"nuclear envelope","supporting_discovery_ids":[21]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,2,13,15,16,17]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[6,9]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[14,23]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[26,32]}],"complexes":["RGS14·Gαi1·H-Ras ternary complex","RGS14·H-Ras·Raf·MEK·ERK complex","NPT2A–NHERF1 complex (regulated by RGS14)"],"partners":["GNAI1","GNAI3","HRAS","RAF1","YWHAG","NHERF1","CAMK2","XPO1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O43566","full_name":"Regulator of G-protein signaling 14","aliases":[],"length_aa":566,"mass_kda":61.4,"function":"Regulates G protein-coupled receptor signaling cascades. Inhibits signal transduction by increasing the GTPase activity of G protein alpha subunits, thereby driving them into their inactive GDP-bound form. Besides, modulates signal transduction via G protein alpha subunits by functioning as a GDP-dissociation inhibitor (GDI). Has GDI activity on G(i) alpha subunits GNAI1 and GNAI3, but not on GNAI2 and G(o)-alpha subunit GNAO1. Has GAP activity on GNAI0, GNAI2 and GNAI3. May act as a scaffold integrating G protein and Ras/Raf MAPkinase signaling pathways. Inhibits platelet-derived growth factor (PDGF)-stimulated ERK1/ERK2 phosphorylation; a process depending on its interaction with HRAS and that is reversed by G(i) alpha subunit GNAI1. Acts as a positive modulator of microtubule polymerisation and spindle organization through a G(i)-alpha-dependent mechanism. Plays a role in cell division. Required for the nerve growth factor (NGF)-mediated neurite outgrowth. Involved in stress resistance. May be involved in visual memory processing capacity and hippocampal-based learning and memory","subcellular_location":"Nucleus; Nucleus, PML body; Cytoplasm; Membrane; Cell membrane; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Cytoplasm, cytoskeleton, spindle; Cytoplasm, cytoskeleton, spindle pole; Cell projection, dendrite; Cell projection, dendritic spine; Postsynaptic density","url":"https://www.uniprot.org/uniprotkb/O43566/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RGS14","classification":"Not Classified","n_dependent_lines":6,"n_total_lines":1208,"dependency_fraction":0.004966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RGS14","total_profiled":1310},"omim":[{"mim_id":"602514","title":"REGULATOR OF G PROTEIN SIGNALING 16; RGS16","url":"https://www.omim.org/entry/602514"},{"mim_id":"602513","title":"REGULATOR OF G PROTEIN SIGNALING 14; RGS14","url":"https://www.omim.org/entry/602513"},{"mim_id":"602512","title":"REGULATOR OF G PROTEIN SIGNALING 12; RGS12","url":"https://www.omim.org/entry/602512"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Nucleoplasm","reliability":"Uncertain"},{"location":"Plasma membrane","reliability":"Uncertain"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":239.9}],"url":"https://www.proteinatlas.org/search/RGS14"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"O43566","domains":[{"cath_id":"1.10.167.10","chopping":"61-190","consensus_level":"high","plddt":92.8748,"start":61,"end":190},{"cath_id":"3.10.20.90","chopping":"303-372","consensus_level":"high","plddt":88.643,"start":303,"end":372},{"cath_id":"3.10.20.90","chopping":"374-444","consensus_level":"high","plddt":85.3713,"start":374,"end":444}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O43566","model_url":"https://alphafold.ebi.ac.uk/files/AF-O43566-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O43566-F1-predicted_aligned_error_v6.png","plddt_mean":67.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RGS14","jax_strain_url":"https://www.jax.org/strain/search?query=RGS14"},"sequence":{"accession":"O43566","fasta_url":"https://rest.uniprot.org/uniprotkb/O43566.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O43566/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O43566"}},"corpus_meta":[{"pmid":"11387333","id":"PMC_11387333","title":"RGS12 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This is distinct from the N-terminal RGS box, which confers GTPase-accelerating protein (GAP) activity on Gαi/o subunits, establishing that RGS14 has two independent Gα interaction sites with different biochemical activities.\",\n      \"method\": \"GTPγS binding assays, intrinsic tryptophan fluorescence assays, in vitro binding experiments with purified proteins\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple in vitro assays (GTPγS binding, fluorescence) with recombinant proteins; replicated across RGS12 and RGS14; foundational biochemical characterization\",\n      \"pmids\": [\"11387333\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"RGS14 binds activated Rap1 and Rap2 (but not Ras) through a domain distinct from the RGS domain that shares sequence identity with the Ras/Rap binding domain of B-Raf and Raf-1 kinases. RGS14 preferentially stimulates GTPase activity of Gαo over Gαi1 in brain membranes and in reconstitution assays.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding with Rap2 mutants, GTPase assays with brain membranes, deletion analysis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — yeast two-hybrid plus in vitro binding and enzymatic assays in a single study; multiple orthogonal methods\",\n      \"pmids\": [\"10926822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"RGS14 acts as a GAP for Gαi subfamily members and attenuates IL-8 receptor-mediated MAPK activation. RGS14 does not exhibit GAP activity toward Gαs or Gαq. Although not a GAP for G12/13α, RGS14 impairs G13α-mediated c-fos SRE activation. An RGS14 mutant (EN92/93AA) defective in Gαi signaling inhibition still blocks SRE activation. RGS14 localizes predominantly to the cytosol but is recruited to membranes by constitutively active G13αQ226L.\",\n      \"method\": \"GTPase assays, cell-based signaling reporter assays, subcellular fractionation, dominant-negative mutant analysis\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-based assays plus biochemical GAP assays; single lab but multiple readouts\",\n      \"pmids\": [\"10953050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Full-length RGS14 in brain exists in two immunoreactive forms (~60 kDa and ~55 kDa) in cytosol and two membrane subpopulations. Recombinant RGS14 is a non-selective GAP for Gαi1 and Gαo, with full-length RGS14 approximately 10-fold more potent than the isolated RGS domain alone. GDI activity toward Gαi (but not Gαo) is restricted to the C-terminus containing the GoLoco domain, and the isolated RBD domain does not act as a GAP for Rap1.\",\n      \"method\": \"Immunoblotting with affinity-purified antisera, reconstitution GTPase assays, pulldown from brain membranes\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution GTPase assays plus domain mapping with isolated fragments; multiple orthogonal approaches in one study\",\n      \"pmids\": [\"11739605\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PKA phosphorylates RGS14 at two specific sites in response to cAMP elevation; one site, threonine 494 (Thr494), is immediately adjacent to the GoLoco motif. Mimicking phosphorylation at Thr494 enhanced GDI activity of RGS14 toward Gαi nearly 3-fold without affecting GAP activity toward Gαi or Gαo.\",\n      \"method\": \"Radiolabeled phosphate incorporation, PKA inhibitor experiments, phosphomimetic mutant biochemical assays, cAMP stimulation in cells\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assays plus phosphomimetic mutagenesis with functional readout; multiple methods in single study\",\n      \"pmids\": [\"12534294\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The RGS14 GoLoco domain discriminates among Gαi isoforms: it inhibits guanine nucleotide exchange on Gαi1 and Gαi3, but not Gαi2. Gαi2 could be rendered sensitive to RGS14 GDI activity by replacing residues within the αA/αB and αB/αC loops of the α-helical domain, identifying these loops as key selectivity determinants.\",\n      \"method\": \"In vitro GTPγS binding assays, Gαi2 chimeric mutant analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical assays with mutagenesis identifying structural determinants of selectivity\",\n      \"pmids\": [\"15337739\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"RGS14 is expressed in mouse embryos immediately prior to the first mitosis, colocalizes with the anastral mitotic apparatus of the mouse zygote, and is essential for completion of the first mitotic division. Loss of Rgs14 in the zygote causes cytofragmentation and failure to progress to the 2-cell stage. During interphase in proliferating cells RGS14 segregates to the nucleus, and during mitosis it localizes to the mitotic spindle and centrioles. Altering RGS14 levels in proliferating cells causes growth arrest.\",\n      \"method\": \"Loss-of-function in mouse embryos, colocalization imaging, overexpression/knockdown in proliferating cells\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic loss-of-function with clear cellular phenotype, direct localization imaging; replicated in multiple experimental systems\",\n      \"pmids\": [\"15525537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The RGS and GoLoco domains of RGS14 are independently able to inhibit signaling downstream of Gi via the βγ pathway. Targeting the isolated GoLoco domain to membranes (by myristoylation/palmitoylation or Rap binding) enhances its inhibitory activity. In the context of full-length RGS14, the RGS and GoLoco domains cooperate to confer maximal inhibition of Gi signaling.\",\n      \"method\": \"Cell-based signaling assays in HEK-m2 cells with domain mutants and truncation constructs, myristoylation targeting constructs\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-based functional assays with domain mutants and multiple constructs; single lab\",\n      \"pmids\": [\"15112653\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"RGS14 localizes to centrosomes via its first Ras-binding domain, and shuttles between cytoplasm and nucleus via CRM-1-dependent nuclear export. Mutation of the nuclear export signal or leptomycin B treatment causes nuclear accumulation and association with PML nuclear bodies. A mutant defective in nuclear export also fails to target to centrosomes, indicating that nucleocytoplasmic shuttling is required for centrosomal localization.\",\n      \"method\": \"Immunofluorescence, leptomycin B treatment, NES mutant analysis, domain deletion constructs\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiments with domain mapping and pharmacological perturbation; single lab\",\n      \"pmids\": [\"15520006\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"RGS14 is a microtubule-associated protein that binds directly to polymerized (but not depolymerized) microtubules with Kd ~1.3 μM. RGS14 copurifies with tubulin through multiple polymerization/depolymerization cycles from brain. Both RGS14 and Gαi1-GTP promote tubulin polymerization in the presence of microtubule-associated proteins, but preincubation of RGS14 with Gαi1-GDP abolishes this activity. Depletion of RGS14 from cell extracts blocks mitotic aster formation in vitro.\",\n      \"method\": \"Microtubule co-sedimentation, direct binding to pure tubulin with Kd measurement, brain extract purification, in vitro aster formation assay, RGS14 depletion\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified proteins, direct binding Kd measurement, functional depletion assay; multiple orthogonal methods\",\n      \"pmids\": [\"15917656\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The RBD/GoLoco region of RGS14 (lacking the RGS domain) binds Gαo-GDP, Gαi-GDP, and Gβγ in brain pull-down assays. In reconstituted receptor-G protein systems, this region potentiates RGS4 GAP activity by increasing the apparent affinity of RGS4 for Gαi and Gαo, an activity requiring membranes and/or receptors.\",\n      \"method\": \"Brain pull-down assays, reconstituted M2 receptor/G-protein GTPase assays, steady-state GTPase measurements\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro biochemical assays with brain membranes and reconstitution; multiple activity measurements in single lab\",\n      \"pmids\": [\"15807543\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"RGS14 subcellular localization in HeLa cells is controlled by distinct domains: localization to the nucleus requires the RGS and RBD domains; translocation out of the nucleus depends on the GoLoco/GPR domain; localization to centrosomes depends on the RBD domain. RGS14 binds directly to both inactive (GDP) and active (GTP) forms of Gαi1 and Gαi3, but not Gαi2, and binding to inactive Gαi1/3 at the plasma membrane prevents nuclear or centrosomal association.\",\n      \"method\": \"Confocal imaging of GFP-tagged constructs with domain mutations, co-immunoprecipitation with Gαi isoforms from cells\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct imaging with domain mutants plus co-IP with different Gα isoforms; single lab, multiple readouts\",\n      \"pmids\": [\"16870394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"RGS14 GAP activity and GDI activity toward heterotrimeric G proteins are unaffected by Rap1/Rap2 binding. A heterotrimer composed of N-myristoylated Gαi1 and prenylated Gβγ is resistant to the GDI activity of the RGS14 GoLoco domain, suggesting RGS14 alone cannot dissociate intact heterotrimers.\",\n      \"method\": \"In vitro GTPase assays, GDI assays with assembled heterotrimer, Rap-binding competition experiments\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical assays; negative finding confirmed across multiple assay conditions in single lab\",\n      \"pmids\": [\"16246175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In cellular contexts, RGS14 selectively binds activated H-Ras-GTP (not Rap isoforms), assembles a multiprotein complex with components of the ERK MAPK pathway (Raf, MEK, ERK) in an H-Ras-GTP-dependent manner, and promotes sustained ERK activation. siRNA knockdown of RGS14 inhibited NGF- and bFGF-mediated neuronal differentiation of PC12 cells, a process dependent on Ras-ERK signaling.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, PC12 neurite outgrowth assays, ERK phosphorylation assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP and loss-of-function with specific cellular phenotype; single lab, multiple methods\",\n      \"pmids\": [\"19319189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"RGS14 is highly enriched in CA2 pyramidal neurons and suppresses long-term potentiation (LTP) at Schaffer collateral synapses. Deletion of RGS14 (exons 2–7 knockout) enables robust LTP at CA2 synapses without affecting CA1 plasticity. MEK inhibition blocks LTP in RGS14-deficient CA2 neurons, implicating ERK/MAP kinase signaling. RGS14 KO mice show enhanced spatial learning and object recognition memory without deficits in non-hippocampal tasks.\",\n      \"method\": \"Genetic knockout mice, electrophysiology (LTP recordings), MEK inhibitor pharmacology, behavioral testing\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with defined LTP phenotype, pharmacological rescue with MEK inhibitor, behavioral validation; multiple orthogonal methods\",\n      \"pmids\": [\"20837545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Activated H-Ras binds RGS14 at the R1 Ras-binding domain (RBD) to form a stable complex at cell membranes. RGS14 co-localizes with and forms a complex with Raf kinases in cells, and the regulatory region of Raf-1 binds the RBD region of RGS14. H-Ras and Raf facilitate one another's binding to RGS14. RGS14 selectively inhibits PDGF- but not EGF- or serum-stimulated ERK phosphorylation, dependent on H-Ras binding. Gαi1 binding to RGS14 is mutually exclusive with Raf binding, and Gαi1 recruits RGS14 to the plasma membrane to reverse inhibition of ERK signaling.\",\n      \"method\": \"Co-immunoprecipitation, subcellular localization imaging, ERK phosphorylation assays, domain mutant analysis\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus functional signaling assays; single lab, multiple methods\",\n      \"pmids\": [\"19878719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"RGS14 forms a Gαi/o-dependent complex with the α2A-adrenergic receptor (α2A-AR) in live cells, as measured by BRET. Receptor agonist (UK14304) stimulation disrupts the RGS14·α2A-AR complex while preserving the RGS14·Gαi1 complex. Ric-8A (a non-receptor GEF) facilitates dissociation of both the RGS14·Gαi1 complex and the Gαi1-dependent RGS14·α2A-AR complex after receptor activation.\",\n      \"method\": \"Bioluminescence resonance energy transfer (BRET) in live cells, pharmacological receptor stimulation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — BRET in live cells with receptor agonist and genetic perturbations; single lab, single primary method\",\n      \"pmids\": [\"21880739\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"RGS14 preferentially binds activated H-Ras-GTP in live cells, and inactive Gαi1-GDP markedly enhances RGS14 affinity for H-Ras-GTP (but active Gαi1-GTP does not). Active H-Ras-GTP interacts with a native RGS14·Gαi1 complex in brain lysates. Co-expression of RGS14 and Gαi1 in PC12 cells greatly enhances H-Ras-stimulatory effects on neurite outgrowth. Stimulation of α2A-AR induces a conformational change in the Gαi1·RGS14·H-Ras ternary complex.\",\n      \"method\": \"BRET in live cells, co-immunoprecipitation from brain lysates, PC12 neurite outgrowth assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — BRET, brain co-IP, and functional neurite outgrowth assay; multiple orthogonal methods in single study\",\n      \"pmids\": [\"23250758\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The RBD region of RGS14 mediates an intramolecular interaction that enhances GAP activity of the RGS domain while simultaneously inhibiting GDI activity of the GPSM/GoLoco domain. The RBD region also binds members of the B/R4 subfamily of RGS proteins and enhances their effects on GPCR-activated Gi/o proteins.\",\n      \"method\": \"In vitro GTPase/GDI assays with isolated domains and full-length protein, protein-protein binding assays\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro biochemical assays; single lab, novel intramolecular regulatory mechanism\",\n      \"pmids\": [\"23255434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RGS14 forms a stable complex with inactive Gαi1-GDP at the plasma membrane via its GPR motif and is also recruited to the plasma membrane by activated Gαo-AlF4(-) via its RGS domain. RGS14 undergoes allosteric conformational changes upon Gαi1-GDP binding to the GPR motif, as revealed by hydrogen/deuterium exchange mass spectrometry. RGS14 can form a ternary complex with Gαo-AlF4(-) and a Gαi1-GDP mutant (G42R) simultaneously, and a preformed RGS14·Gαi1-GDP complex retains full capacity to stimulate GTPase activity of Gαo-GTP.\",\n      \"method\": \"Size exclusion chromatography, hydrogen/deuterium exchange mass spectrometry (HDX-MS), BRET, biochemical reconstitution, cellular localization assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — HDX-MS structural approach combined with reconstitution of ternary complex and functional GTPase assay; multiple orthogonal methods\",\n      \"pmids\": [\"25666614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"RGS14 acts as a dedicated GAP for Gαo/i signaling and does not prolong Gβγ signaling following receptor activation. Co-expression of RGS14 inhibits free Gβγ release after agonist stimulation and increases the deactivation rate of Gα, consistent with GAP activity. Overexpression of inactive Gαi1 to recruit RGS14 to the plasma membrane did not alter RGS14's GAP activity toward a second Gαo protein, indicating the GPR motif functions independently of the RGS domain for GAP activity.\",\n      \"method\": \"BRET in live cells, GPCR agonist/antagonist kinetics analysis\",\n      \"journal\": \"Pharmacology research & perspectives\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — BRET live cell assays measuring both Gα and Gβγ dynamics; single lab, single method\",\n      \"pmids\": [\"27713821\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Endogenous RGS14 in neuroblastoma (B35) cells localizes to juxtanuclear membranes encircling the nucleus, at nuclear pore complexes on both sides of the nuclear envelope and within intranuclear membrane channels, and within chromatin-rich and chromatin-poor nuclear regions in a cell-cycle-dependent manner. Endogenous RGS14 was absent from the plasma membrane in resting cells but could be trafficked to the plasma membrane from juxtanuclear membranes (derived from ER/Golgi) following AlF4¯ activation of its G protein binding partners.\",\n      \"method\": \"Confocal imaging, 3D-structured illumination microscopy, AlF4¯ pharmacological activation, subcellular fractionation\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — super-resolution and confocal imaging of endogenous protein; functional trafficking experiment; single lab\",\n      \"pmids\": [\"28934222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RGS14 directly interacts with Ca2+/calmodulin (CaM) and is phosphorylated by CaMKII in vitro. RGS14 associates with CaMKII and CaM in hippocampal CA2 neurons in vivo. These interactions were identified via immunoprecipitation-mass spectrometry proteomics of endogenous RGS14 from mouse brain, validated by direct biochemical binding assays.\",\n      \"method\": \"Immunoprecipitation-mass spectrometry from mouse brain, direct binding assays, in vitro CaMKII phosphorylation assay, confocal colocalization\",\n      \"journal\": \"Journal of proteome research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomics discovery validated by in vitro assays and co-IP; multiple methods in single lab\",\n      \"pmids\": [\"29518331\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RGS14 suppresses plasticity in hippocampal area CA2 by restricting calcium (Ca2+) elevations in dendritic spines. Genetic KO of RGS14 results in nascent LTP requiring NMDA receptors, CaMK, and PKA. WT CA2 neurons display significantly attenuated spine Ca2+ transients during structural plasticity induction compared with RGS14 KO CA2 or CA1 controls. Acute overexpression of RGS14 is sufficient to block spine structural plasticity, and elevated extracellular Ca2+ restores plasticity to RGS14-expressing neurons.\",\n      \"method\": \"Genetic KO mice, two-photon calcium imaging in dendritic spines, pharmacological inhibition (NMDA receptor, CaMK, PKA antagonists), overexpression experiments\",\n      \"journal\": \"eNeuro\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct spine Ca2+ imaging combined with KO and OE experiments plus pharmacological dissection; multiple orthogonal methods\",\n      \"pmids\": [\"29911178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"14-3-3γ directly binds RGS14 at two distinct sites: one phosphorylation-independent site and one phosphorylation-dependent site at Ser-218, potentiated by active H-Ras signaling. The pSer-218-dependent interaction inhibits active Gαi1-AlF4- binding to the RGS domain but does not affect H-Ras or inactive Gαi1-GDP binding. The phosphorylation-independent 14-3-3 interaction inhibits RGS14 nuclear import/nucleocytoplasmic shuttling without affecting Gαi interactions.\",\n      \"method\": \"BRET in live cells, direct binding assays, site-directed mutagenesis at Ser-218\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — BRET plus mutagenesis identifying two functionally distinct binding sites; single lab, multiple readouts\",\n      \"pmids\": [\"30093406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Human genetic variants L505R (LR) and R507Q (RQ) in the nuclear export sequence of RGS14 disrupt RGS14 binding to Gαi1-GDP and to Exportin 1 (XPO1), disturb nucleocytoplasmic equilibrium, and abolish RGS14's capacity to inhibit LTP. Variant LR accumulates irreversibly in the nucleus, preventing membrane localization to dendritic spines. When introduced into mice by CRISPR/Cas9, RGS14-LR is predominantly nuclear in neurons of hippocampus, amygdala, piriform cortex, and striatum. Unlike complete RGS14 KO, LR mice show normal spatial learning, suggesting distinct nuclear vs. dendritic spine functions.\",\n      \"method\": \"Site-directed mutagenesis, co-immunoprecipitation, electrophysiology (LTP), CRISPR/Cas9 knock-in mice, confocal imaging, behavioral testing\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP for binding partners, LTP electrophysiology, CRISPR knock-in mice, behavioral phenotyping; multiple orthogonal methods in single study\",\n      \"pmids\": [\"33410399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RGS14 possesses a carboxy-terminal PDZ ligand required for binding to NHERF1 scaffold protein (at PDZ2) and thereby regulates NPT2A-mediated renal phosphate transport. RGS14 blocks PTH- and FGF23-induced dissociation of the NPT2A-NHERF1 complex and thereby suppresses hormone-sensitive (but not basal) phosphate uptake. RGS14 genetic variants bearing mutations in the PDZ ligand disrupt NHERF1 binding and PTH-sensitive phosphate transport. RGS14 does not affect PTH-directed G protein activation or cAMP formation, implying a post-receptor site of action.\",\n      \"method\": \"Isotope uptake measurements, BRET assays, siRNA knockdown, pull-down and overlay assays, molecular modeling, direct binding assays in proximal tubule cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (isotope transport, BRET, pull-down, mutagenesis, siRNA) in single study\",\n      \"pmids\": [\"35307350\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"RGS14 associates with mu-opioid receptors (MOR) in periaqueductal gray (PAG) neurons. When RGS14 is silenced, morphine increases serine 375 phosphorylation of MOR (a GRK substrate), leading to receptor internalization and recycling and resensitization to morphine with reduced tolerance. RGS14 prevents GRKs from phosphorylating MOR residues required for β-arrestin-mediated endocytosis. Morphine (but not DAMGO) also triggers CaMKII activation in PAG neurons via a mechanism requiring RGS14.\",\n      \"method\": \"siRNA knockdown in vivo, receptor phosphorylation assays, internalization/recycling assays, behavioral antinociception measurement\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo knockdown with mechanistic readouts of receptor phosphorylation and internalization; single lab\",\n      \"pmids\": [\"17825524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RGS14 regulates αMβ2 integrin activation during phagocytosis in macrophages. Exogenous RGS14 expression in COS-7 cells increases binding of C3bi-opsonized targets. Knockdown of RGS14 in J774.A1 macrophages decreases association with C3bi-opsonized particles. This function requires the R333 residue of the RGS14 RBD and the F754 residue of β2, indicating regulation occurs through the Rap/RBD axis.\",\n      \"method\": \"Exogenous expression, siRNA knockdown, binding assays with opsonized particles, point mutant analysis\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function experiments with mutant analysis; single lab, multiple cell types\",\n      \"pmids\": [\"23805333\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RGS14 localizes to mitochondria in hippocampal CA2 pyramidal cells and reduces mitochondrial respiration in vitro. Loss of RGS14 dramatically increases 3-nitrotyrosine levels (oxidative stress marker) in CA2 cells, which is exacerbated following kainic acid-induced status epilepticus and correlates with a lack of SOD2 induction. RGS14 KO mice show accelerated onset of limbic motor seizures and increased mortality. RGS14 KO also results in absence of microgliosis in CA1 and CA2 following seizure.\",\n      \"method\": \"Mitochondrial localization imaging, in vitro mitochondrial respiration assay, kainic acid seizure model in KO mice, proteomics, immunostaining for oxidative stress markers\",\n      \"journal\": \"Neurobiology of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mice with defined seizure phenotype, localization experiment, in vitro functional assay; single lab, multiple methods\",\n      \"pmids\": [\"37075948\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Small-molecule inhibitors targeting the solvent-exposed 'canyon' in the RGS14 RGS-box that interacts with the Gα switch I region can non-covalently and selectively inhibit RGS14 GAP activity. Two second-generation analogs (Z55660043 and Z55627844) inhibited RGS14 GAP activity in both fluorescence-based and radioactive GTP hydrolysis assays without measurable cytotoxicity. One analog showed favorable in vivo pharmacokinetics and CNS penetration.\",\n      \"method\": \"Structure-guided virtual screening, fluorescence-based GAP assays, radioactive GTP hydrolysis assays, cytotoxicity assays, in vivo pharmacokinetics\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro GAP assays with confirmed inhibition by two orthogonal methods; preprint, single lab, no structural validation yet\",\n      \"pmids\": [\"bio_10.1101_2025.06.11.659181\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RGS14 physically interacts with GNAI3 (Gαi3) in spermatogonial stem cells (SSCs), as shown by co-immunoprecipitation. RGS14 knockdown suppresses SSC proliferation, induces apoptosis, inhibits PLPP2 expression, and reduces MAPK signaling activation. Overexpression of PLPP2 rescues phenotypic defects caused by RGS14 depletion, identifying a RGS14–GNAI3–PLPP2 regulatory axis.\",\n      \"method\": \"Single-cell RNA sequencing, siRNA knockdown, co-immunoprecipitation, RNA sequencing, PLPP2 rescue overexpression, proliferation/apoptosis assays\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus loss-of-function with rescue experiment; single lab, multiple methods\",\n      \"pmids\": [\"40352663\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RGS14 regulates hormone-sensitive renal phosphate transport through a linker region between the RGS and RBD1 domains: Ser266 and Ser269 within this α-helical linker are phosphorylated in response to PTH and FGF23. Ser266Ala/Ser269Ala substitutions abolish RGS14 regulatory effects on hormone-sensitive phosphate transport while an intact PDZ ligand is also required. Truncation constructs lacking the RGS domain are fully functional for phosphate transport regulation.\",\n      \"method\": \"RGS14 truncation constructs, phosphorylation assays with PTH and FGF23, phosphate uptake measurements, alanine mutagenesis of Ser266/Ser269\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis combined with functional transport assays and direct phosphorylation measurement; single lab\",\n      \"pmids\": [\"39792018\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RGS14 is a multifunctional scaffolding protein that integrates heterotrimeric G protein and Ras/Raf/ERK MAPK signaling: its N-terminal RGS domain acts as a GAP for Gαi/o-GTP, its C-terminal GoLoco/GPR motif binds inactive Gαi1/3-GDP to exert GDI activity (potentiated by PKA phosphorylation at Thr494), and its tandem Ras-binding domains selectively bind activated H-Ras-GTP in cells to scaffold a Ras–Raf–MEK–ERK complex; inactive Gαi1-GDP binding enhances H-Ras affinity and recruits the full complex to the plasma membrane while displacing Raf, allowing GPCRs to dynamically regulate the complex; in hippocampal CA2 neurons RGS14 suppresses LTP and learning by restricting spine Ca2+ elevations and downstream CaMK/PKA/ERK signaling; RGS14 also localizes to mitochondria, centrosomes, the mitotic spindle, and the nucleus (via CRM1-dependent export), directly binds polymerized microtubules to promote spindle assembly, interacts with 14-3-3γ to regulate both its Gα-binding and nuclear import functions, and regulates renal phosphate homeostasis through a PDZ-ligand/NHERF1/NPT2A axis that is controlled by phosphorylation of linker-region serines in response to PTH and FGF23.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RGS14 is a multidomain scaffolding protein that integrates heterotrimeric G protein signaling with Ras/Raf/ERK MAPK signaling and microtubule-based mitotic functions [#0, #13]. Its N-terminal RGS domain accelerates GTP hydrolysis on Gαi/o subunits, while a C-terminal GoLoco/GPR motif binds Gαi1 and Gαi3 (but not Gαi2) selectively in their GDP-bound state and acts as a guanine nucleotide dissociation inhibitor, establishing two independent, biochemically distinct Gα interaction sites [#0, #3, #5]. These activities are regulated post-translationally: PKA phosphorylation at Thr494 adjacent to the GoLoco motif enhances GDI activity ~3-fold, and an intramolecular interaction mediated by the Ras-binding region enhances GAP activity while inhibiting GDI activity [#4, #18]. Through its tandem Ras-binding domains RGS14 selectively binds activated H-Ras-GTP in cells and assembles an H-Ras-dependent Raf–MEK–ERK complex to promote sustained ERK activation and Ras-dependent neuronal differentiation, with inactive Gαi1-GDP markedly enhancing H-Ras affinity and Gαi1 binding being mutually exclusive with Raf binding, allowing GPCRs to dynamically tune the complex [#13, #15, #17]. RGS14 shuttles between cytoplasm and nucleus via CRM1/XPO1-dependent export and localizes to centrosomes, the mitotic spindle, and microtubules, binding polymerized microtubules directly to promote spindle/aster assembly and being required for the first embryonic mitotic division [#6, #8, #9]. In hippocampal CA2 pyramidal neurons RGS14 suppresses long-term potentiation and spatial learning by restricting dendritic spine Ca2+ elevations and downstream NMDA-receptor/CaMK/PKA/ERK signaling [#14, #23]. RGS14 also regulates hormone-sensitive renal phosphate transport through a C-terminal PDZ ligand that binds NHERF1 to stabilize the NPT2A–NHERF1 complex against PTH/FGF23-induced dissociation, an activity controlled by phosphorylation of linker serines Ser266/Ser269 [#26, #32]. Human variants in the RGS14 nuclear export sequence (L505R, R507Q) disrupt Gαi1 and XPO1 binding, trap the protein in the nucleus, and abolish its capacity to inhibit LTP [#25].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established that RGS14 acts on G protein signaling through a GAP activity selective for the Gαi/o subfamily and through a domain binding activated Rap1/Rap2, defining it as more than a simple GTPase regulator.\",\n      \"evidence\": \"GTPase assays with brain membranes, yeast two-hybrid, and cell-based SRE reporter assays\",\n      \"pmids\": [\"10926822\", \"10953050\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve whether Rap binding is functional in cells\", \"Mechanism linking RGS14 to MAPK attenuation unclear\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Resolved that RGS14 carries two independent Gα interaction sites with opposite biochemical activities — an RGS-box GAP and a GoLoco-motif GDI selective for GDP-bound Gαi1/2/3 — defining its bifunctional regulation of the Gαi cycle.\",\n      \"evidence\": \"GTPγS binding and intrinsic tryptophan fluorescence assays with purified proteins, plus reconstitution GTPase assays and domain mapping\",\n      \"pmids\": [\"11387333\", \"11739605\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular consequences of dual GAP/GDI activity not addressed\", \"No structural model of the two binding modes\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Showed that PKA phosphorylation at Thr494 adjacent to the GoLoco motif selectively potentiates GDI activity, providing a mechanism by which cAMP signaling tunes RGS14's effect on Gαi.\",\n      \"evidence\": \"In vitro PKA kinase assays, phosphomimetic mutagenesis with GDI/GAP readouts, cAMP stimulation in cells\",\n      \"pmids\": [\"12534294\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of second PKA site not functionally defined\", \"In vivo relevance of Thr494 phosphorylation untested\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Linked RGS14 to cell division and subcellular trafficking, showing it is essential for the first embryonic mitosis, localizes to centrosomes/spindle via its RBD, and shuttles between nucleus and cytoplasm via CRM1-dependent export.\",\n      \"evidence\": \"Loss-of-function in mouse zygotes, colocalization imaging, NES mutagenesis with leptomycin B, domain deletion constructs\",\n      \"pmids\": [\"15525537\", \"15520006\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular role of RGS14 at the spindle not defined\", \"Link between G protein activities and mitotic function unclear\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identified RGS14 as a direct microtubule-associated protein that binds polymerized microtubules and promotes tubulin polymerization and aster formation, providing a biochemical basis for its spindle role.\",\n      \"evidence\": \"Microtubule co-sedimentation, direct Kd measurement with pure tubulin, brain extract co-purification, in vitro aster formation with RGS14 depletion\",\n      \"pmids\": [\"15917656\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Gαi1-GDP inhibition of tubulin polymerization is regulated in vivo unknown\", \"Mitotic phenotype mechanism not connected to MT binding directly\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Mapped the domain logic of RGS14 localization, showing nuclear targeting requires RGS+RBD, nuclear exit requires GoLoco, and centrosome targeting requires the RBD, while inactive Gαi1/3 binding at the membrane sequesters RGS14 away from nucleus/centrosome.\",\n      \"evidence\": \"Confocal imaging of GFP-tagged domain mutants and co-IP with Gαi isoforms\",\n      \"pmids\": [\"16870394\", \"16246175\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab imaging not independently confirmed\", \"Whether RGS14 dissociates intact heterotrimers in cells unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Established RGS14 as a scaffold for the Ras–Raf–MEK–ERK pathway, showing it selectively binds H-Ras-GTP in cells, assembles an ERK-pathway complex, and is required for Ras-dependent neuronal differentiation.\",\n      \"evidence\": \"Co-IP, siRNA knockdown, PC12 neurite outgrowth and ERK phosphorylation assays\",\n      \"pmids\": [\"19319189\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs. indirect Raf/MEK/ERK binding not fully dissected\", \"Single cellular system\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined the architecture and regulation of the RGS14–H-Ras–Raf complex, showing H-Ras binds the R1 RBD, H-Ras and Raf cooperatively bind RGS14, and Gαi1 binding is mutually exclusive with Raf, letting Gαi1 reverse RGS14's ERK inhibition by membrane recruitment; in parallel, in vivo work revealed RGS14 suppresses CA2 LTP and learning via ERK.\",\n      \"evidence\": \"Co-IP, localization imaging, ERK assays with domain mutants; and RGS14 KO mice with LTP electrophysiology, MEK inhibitor rescue, and behavioral testing\",\n      \"pmids\": [\"19878719\", \"20837545\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How GPCR input switches the complex in neurons not directly shown\", \"CA2-specific molecular targets of RGS14 not fully defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated that RGS14 forms a Gαi/o-dependent complex with the α2A-adrenergic receptor that is disrupted by agonist and by the GEF Ric-8A, placing RGS14 in a receptor-coupled signaling module.\",\n      \"evidence\": \"BRET in live cells with pharmacological receptor stimulation and Ric-8A co-expression\",\n      \"pmids\": [\"21880739\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs. Gα-bridged receptor contact not distinguished\", \"Single primary method\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed that inactive Gαi1-GDP allosterically enhances RGS14 affinity for H-Ras-GTP and that a native Gαi1·RGS14·H-Ras ternary complex exists in brain and is conformationally responsive to α2A-AR stimulation, unifying G protein and Ras inputs on one scaffold.\",\n      \"evidence\": \"BRET in live cells, co-IP from brain lysates, PC12 neurite outgrowth assays\",\n      \"pmids\": [\"23250758\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Gαi-enhanced H-Ras binding unknown\", \"Downstream output of the ternary complex in neurons not measured\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Revealed an intramolecular RBD-mediated regulation that reciprocally enhances RGS-domain GAP activity while inhibiting GoLoco GDI activity, and showed RGS14 binds and potentiates B/R4 RGS proteins, adding autoregulatory and cooperative layers.\",\n      \"evidence\": \"In vitro GTPase/GDI assays with isolated and full-length constructs and protein-protein binding assays; and macrophage integrin/phagocytosis assays\",\n      \"pmids\": [\"23255434\", \"23805333\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo significance of intramolecular regulation untested\", \"Phagocytosis role from single lab\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Captured the conformational logic of G protein engagement, showing GPR-bound Gαi1-GDP induces allosteric changes and that RGS14 can simultaneously bind Gαi1-GDP (GPR) and Gαo (RGS) while retaining GAP activity, establishing functional independence of the two Gα sites.\",\n      \"evidence\": \"Size exclusion chromatography, HDX-MS, BRET, biochemical reconstitution of the ternary complex with GTPase assay\",\n      \"pmids\": [\"25666614\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No high-resolution crystal/cryo-EM structure\", \"How allostery couples to Ras/ERK scaffolding not shown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Localized endogenous RGS14 to juxtanuclear ER/Golgi-derived membranes, nuclear pore complexes, and intranuclear channels, and showed it can traffic to the plasma membrane upon G protein activation, refining its dynamic localization in neuronal cells.\",\n      \"evidence\": \"Confocal and 3D-SIM super-resolution imaging of endogenous protein with AlF4⁻ activation and fractionation\",\n      \"pmids\": [\"28934222\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional role at nuclear pores undefined\", \"Single-lab imaging\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Connected RGS14 to Ca2+/CaMKII signaling and 14-3-3γ regulation, showing RGS14 binds CaM, is a CaMKII substrate, and is regulated by dual 14-3-3γ sites that separately control Gαi binding and nuclear shuttling, while its CA2 LTP suppression operates by limiting spine Ca2+.\",\n      \"evidence\": \"IP-mass spectrometry from brain, in vitro binding and CaMKII phosphorylation, BRET, Ser-218 mutagenesis; and two-photon spine Ca2+ imaging with KO/OE and pharmacology\",\n      \"pmids\": [\"29518331\", \"29911178\", \"30093406\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CaMKII phosphorylation feeds back on RGS14 function not resolved\", \"Link between 14-3-3γ regulation and spine Ca2+ control untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated that human RGS14 NES variants (L505R, R507Q) disrupt Gαi1 and XPO1 binding, trap RGS14 in the nucleus, and abolish LTP inhibition, while leaving spatial learning intact, dissociating RGS14's nuclear from dendritic-spine functions.\",\n      \"evidence\": \"Site-directed mutagenesis, co-IP, LTP electrophysiology, CRISPR/Cas9 knock-in mice, imaging, behavior\",\n      \"pmids\": [\"33410399\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nuclear function of RGS14 not mechanistically defined\", \"Disease association of variants not established\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Established a non-canonical, post-receptor renal function in which RGS14's C-terminal PDZ ligand binds NHERF1 to stabilize the NPT2A–NHERF1 complex and suppress PTH/FGF23-sensitive phosphate uptake, independent of G protein/cAMP signaling.\",\n      \"evidence\": \"Isotope phosphate uptake, BRET, siRNA, pull-down/overlay assays, mutagenesis in proximal tubule cells\",\n      \"pmids\": [\"35307350\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo whole-animal phosphate phenotype not shown\", \"Whether other RGS14 domains modulate this axis unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified a mitochondrial role for RGS14 in CA2 neurons, where it reduces respiration and limits oxidative stress, with KO mice showing accelerated seizures and increased mortality, linking RGS14 to neuronal metabolic and neuroprotective functions.\",\n      \"evidence\": \"Mitochondrial localization imaging, respiration assays, kainic acid seizure model in KO mice, proteomics, oxidative stress immunostaining\",\n      \"pmids\": [\"37075948\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism of respiration control unknown\", \"Whether mitochondrial role depends on G protein/Ras activities unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Refined the renal mechanism and extended RGS14 into spermatogonial stem cell biology and pharmacology: linker serines Ser266/Ser269 are PTH/FGF23-phosphorylated and required for phosphate-transport regulation; RGS14 binds GNAI3 to drive an SSC proliferation/MAPK/PLPP2 axis; and selective small-molecule RGS-box inhibitors were developed.\",\n      \"evidence\": \"Truncation/alanine mutagenesis with phosphate transport and phosphorylation assays; co-IP, siRNA, RNA-seq and PLPP2 rescue in SSCs; structure-guided GAP-inhibitor screening (one preprint)\",\n      \"pmids\": [\"39792018\", \"40352663\", \"bio_10.1101_2025.06.11.659181\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Kinase responsible for Ser266/Ser269 phosphorylation not identified\", \"Inhibitors lack structural validation and in vivo efficacy data\", \"SSC axis from single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RGS14's distinct activities — Gαi/o GAP/GDI, H-Ras/ERK scaffolding, microtubule binding, mitochondrial regulation, nuclear shuttling, and renal NHERF1/NPT2A control — are coordinately switched within a single cell, and what defines the nuclear function, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated structural model coupling the multiple domains\", \"Nuclear function of RGS14 undefined\", \"Physiological switch governing which activity dominates in a given cell type unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 5, 12, 18, 20]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [13, 15, 17, 26]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [9, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 3, 11]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [11, 15, 19, 21]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6, 8, 11, 21, 25]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [6, 8, 11]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [29]},\n      {\"term_id\": \"GO:0005635\", \"supporting_discovery_ids\": [21]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 2, 13, 15, 16, 17]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [6, 9]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [14, 23]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [26, 32]}\n    ],\n    \"complexes\": [\n      \"RGS14·Gαi1·H-Ras ternary complex\",\n      \"RGS14·H-Ras·Raf·MEK·ERK complex\",\n      \"NPT2A–NHERF1 complex (regulated by RGS14)\"\n    ],\n    \"partners\": [\n      \"GNAI1\",\n      \"GNAI3\",\n      \"HRAS\",\n      \"RAF1\",\n      \"YWHAG\",\n      \"NHERF1\",\n      \"CAMK2\",\n      \"XPO1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}